Gata6 Potently Initiates Reprograming of Pluripotent and Differentiated Cells to Extraembryonic Endoderm Stem Cells

Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells

Sissy E. Wamaitha,1 Ignacio del Valle,1 Lily T.Y. Cho,1,4 Yingying Wei,2,5 Norah M.E. Fogarty,1 Paul Blakeley,1 Richard I. Sherwood,3 Hongkai Ji,2 and Kathy K. Niakan1 1Mill Hill Laboratory, The Francis Crick Institute, London NW7 1AA, United Kingdom; 2Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205, USA; 3Brigham and Women’s Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types. [Keywords: reprograming; pluripotency; mouse embryonic stem cells; human embryonic stem cells; extraembryonic endoderm; Gata6] Supplemental material is available for this article. Received December 9, 2014; revised version accepted May 21, 2015.

One of the earliest specification events during mammali- et al. 2006; van den Berg et al. 2008; Zhang et al. 2008; an development is the divergence of pluripotent epiblast Hall et al. 2009; Niwa et al. 2009; Festuccia et al. 2012). progenitor (EPI) cells, which give rise to the embryo prop- In contrast, PrE and self-renewing XEN cells lack ex- er, and primitive endoderm (PrE) cells, which mainly con- pression of pluripotency genes and require an extraembry- tribute to the yolk sac. In mice, embryonic stem (ES) cells onic endoderm (ExEn) program, including transcription and extraembryonic endoderm stem (XEN) cells can be de- factors Gata4, Gata6, Sox7, and Sox17 (Soudais et al. rived from the EPI and PrE lineages, respectively, and re- 1995; Arman et al. 1998; Morrisey et al. 1998; Koutsoura- tain characteristics of their cell type of origin (Evans and kis et al. 1999; Futaki et al. 2004; Capo-Chichi et al. 2005; Kaufman 1981; Martin 1981; Kunath et al. 2005). Mouse Kunath et al. 2005; Chazaud et al. 2006; Shimoda et al. ES (mES) cells are able to self-renew and remain pluripo- 2007; Brown et al. 2010b; Morris et al. 2010; Niakan tent and depend on a gene regulatory network surround- et al. 2010; Artus et al. 2011; Schrode et al. 2014). It re- ing the core transcription factors Oct4, Sox2, and Nanog mains unclear how cells within the early embryo diverge (Boyer et al. 2005; Loh et al. 2006; Chen et al. 2008). Tran- to favor either a pluripotency or an ExEn gene regulatory scription factors such as Esrrb and Klf4 are also regulated network. The overexpression of the zinc finger transcrip- by and reinforce the core pluripotency factors (Ivanova tion factor Gata6 or Gata4 is sufficient to reprogram mES cells into XEN-like cells that contribute to PrE-derived lineages in vivo (Fujikura et al. 2002; Shimosato et al. 2007). The GATA factors are therefore part of a sub- Present addresses: 4Neusentis, Pfizer Worldwide Research and Develop- set of transcription factors that are capable of inducing cel- 5 ment, Great Abington, Cambridge CB21 6GS, UK; Department of Statis- lular reprograming, although precisely how this occurs tics, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. Corresponding author: [email protected] Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.257071. © 2015 Wamaitha et al. This article, published in Genes & Develop- 114. Freely available online through the Genes & Development Open Ac- ment, is available under a Creative Commons License (Attribution 4.0 In- cess option. ternational), as described at http://creativecommons.org/licenses/by/4.0/.

GENES & DEVELOPMENT 29:1239–1255 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/15; www.genesdev.org 1239 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al. has yet to be elucidated. Ectopic expression of the tran- six transcription factors (Gata4, Gata6, Hnf4a, Foxa3, scription factor MyoD converts fibroblasts to myogenic Sox7, and Sox17) that are expressed in the PrE or its deriv- cells (Davis et al. 1987), a combination of transcription atives and are functionally required to establish or main- factors reprograms fibroblasts to induced pluripotent tain this lineage (Chen et al. 1994; Soudais et al. 1995; stem (iPS) cells (Takahashi and Yamanaka 2006), and in Molkentin et al. 1997; Kaestner et al. 1998; Morrisey vivo pancreatic exocrine cells can be reprogramed into in- et al. 1998; Koutsourakis et al. 1999; Capo-Chichi et al. sulin-secreting β-like cells (Zhou et al. 2008). Similarly, 2005; Artus et al. 2011; Schrode et al. 2014). To investigate overexpression of Cdx2 is sufficient to reprogram mES whether their expression is sufficient to induce repro- cells to trophectoderm-like cells that contribute solely graming of mES cells to iXEN cells, we used a site-specific to placental lineages in vivo (Niwa et al. 2005). However, recombination-based integration strategy (Hochedlinger the mechanisms by which single transcription factors re- et al. 2005; Beard et al. 2006) to generate mES cells ex- program existing gene expression patterns toward that of pressing a single copy of a tetracycline/doxycycline-in- their target cell type remain unclear. ducible Gata4, Gata6, Sox7, Sox17, Hnf4a,orFoxa3 Induction of the SRY homeobox gene Sox17 has also transgene. To test the fidelity of the system, we also engi- been shown to induce ExEn gene expression in mES cells neered control mES cells that induce the expression of a (Niakan et al. 2010; McDonald et al. 2014). Intriguingly, gene encoding a red fluorescent protein, dsRed. We con- SOX17 induction in human ES (hES) cells instead drives firmed robust induction above or close to levels present an embryonic endoderm program (Seguin et al. 2008). in embryo-derived XEN (eXEN) cells by quantitative This incongruence is consistent with our previous obser- RT–PCR (qRT–PCR) analysis of the Flag- or V5-tagged vations that the initial ES cell state influences differentia- transgene (Supplemental Fig. S1A). We also observed ro- tion outcomes (Cho et al. 2012). Furthermore, while bust red fluorescence in dsRed-overexpressing mES cells the induction of SOX7 drives ExEn gene expression in (Supplemental Fig. S1B). hES cells (Seguin et al. 2008), stable self-renewing human To determine the effect of these factors under condi- XEN cells have yet to be established. The effect of GATA tions that would otherwise maintain pluripotency, we in- factor induction in hES cells has not been tested, and it is duced exogenous expression with doxycycline for 6 d in unclear whether Gata6 can function as a master transcrip- the presence of LIF and serum, which are known to main- tional regulator to induce a XEN program from cells other tain mES cell self-renewal indefinitely in culture (Fig. 1A). than mES cells. Gata6 or Gata4 overexpression resulted in reprograming We developed a highly efficient approach to understand to cells with the dispersed, refractile, and stellate mor- the molecular mechanisms of Gata6-mediated reprogram- phology characteristic of eXEN (Fig. 1B) and growth fac- ing and show that Gata6 is a potent inducer of lineage re- tor-converted XEN (cXEN) cells (Kunath et al. 2005; Cho programing in multiple cell types. We demonstrate that a et al. 2012). qRT–PCR analysis of the 3′ untranslated re- short pulse of Gata6 induction is ample to perturb gene ex- gion (UTR) confirmed that Gata6-orGata4-induced cells pression in mES cells and initiate conversion to induced expressed endogenous Gata6 and Gata4 as well as key XEN (iXEN) cells, while longer induction fully down-regu- ExEn genes, including Sox7, Sox17, Lama1, Col4a1, lates the pluripotency program. Using genome-wide tran- Sparc, Dab2, Foxa3, and Hnf4a, to levels above or close scriptional and chromatin immunoprecipitation (ChIP) to those present in eXEN cells (Fig. 1C; Supplemental analyses, we found that Gata6 is able to rapidly and Fig. S1C). Immunofluorescence analysis also confirmed directly inhibit core and peripheral genes within the pluri- that both Gata4-induced (data not shown) and Gata6-in- potency regulatory network as well as directly activate an duced cells express Gata4, Gata6, Sox17, and Laminin pro- ExEn program. Despite lingering expression of Oct4 fol- teins (Fig. 1D). Moreover, Gata6-induced cells no longer lowing Gata6 induction, loss-of-function analysis sug- expressed Oct4 protein (Fig. 1D), suggesting that they gests that Oct4 is not required to drive this lineage have been reprogramed to iXEN cells. switch in mES cells. Gata6 expression in more committed In contrast, expression of Sox7, Sox17, Hnf4a,orFoxa3 neural cells also drives reprograming to iXEN-like cells. failed to induce a morphological switch to XEN-like cells We show that GATA6 induction in hES cells initiates within 6 d of induction (Fig. 1A). These factors inconsis- ExEn expression and is sufficient to inhibit core pluripo- tently up-regulated ExEn genes and failed to up-regulate tency gene expression. Our findings have important impli- the expression of factors such as Col4a1, Lama1,or cations for understanding how transcription factors Hnf4a to eXEN cell levels (Fig. 1C; Supplemental Fig. function to drive a cell fate switch and provide fundamen- S1C,D). We and others have previously observed that tal insights into early mammalian cell fate specification. Sox17-overexpressing cells retained mES cell-like morphology and the expression of Pou5f1, Sox2, and Nanog after 48 h of induction (Niakan et al. 2010; McDonald et al. Results 2014). Similarly, Sox17, Sox7,orFoxa3 levels of Pou5f1, Nanog, and Sox2 were comparable with the expression Gata6 or Gata4 expression is uniquely sufficient to induce in dsRed control cells after 6 d of induction (Fig. 1C). rapid reprograming of mES cells to iXEN cells Moreover, Sox7-expressing colonies retain mES cell-like While Gata4 and Gata6 are able to reprogram mES cells, it morphology and persistently express Oct4 protein despite is unclear whether other endoderm transcription factors a few Gata4-, Gata6-, Sox17-, and Laminin-expressing are also able to mediate this cell fate switch. We selected cells at the periphery of colonies (Fig. 1D).

1240 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently reprograms diverse cell types

Figure 1. Gata6 or Gata4 induction is uniquely sufficient to reprogram mES cells to XEN cells. (A) Representative phase-contrast images of dsRed-, Foxa3-, Hnf4a-, Gata4-, Gata6-, Sox7-, or Sox17-induced cells after 6 d of doxycycline treatment in the presence or absence of LIF. Bars, 100 μm. (B) Phase-contrast images of uninduced mES cells and eXEN cells. (C) qRT–PCR analysis for selected pluripotency and endoderm transcripts in dsRed-, Foxa3-, Hnf4a-, Gata4-, Gata6-, Sox7-, or Sox17-induced cells after 6 d of doxycycline treatment in the presence (solid) or absence (checkered) of LIF. Relative expression reflected as fold difference over uninduced mES cells normalized to Gapdh. Data are mean ± SEM of two to three biological replicates and four technical replicates. (∗) P < 0.05; (∗∗) P < 0.01; (∗∗∗) P <0 .001. (D) Immunofluorescence analysis for Gata4, Gata6, Sox17, Laminin, or Oct4 (all green) with DAPI merge (blue) in uninduced mES cells or Sox7-orGata6-overexpressing mES cells after 6 d of doxycycline treatment.

GENES & DEVELOPMENT 1241 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al.

We next induced transgene expression in the absence of by 6 h, followed by Sox2 and Esrrb at 8 and 12 h, respec- LIF to determine whether destabilizing pluripotency via tively. Western blot analysis over the 12-h period con- altered culture conditions would facilitate Sox7, Sox17, firmed these dynamics, with steady down-regulation of Hnf4a,orFoxa3 induction of mES to iXEN cell reprogram- Nanog (Supplemental Fig. S2A). However, Pou5f1 expres- ing within 6 d. Furthermore, as activation of FGF signaling sion was unchanged at these early time points (Fig. 2C). In is required for PrE development and derivation of XEN contrast, endogenous Gata6 and Gata4 were up-regulated cells routinely involves addition of exogenous FGF (Feld- more than twofold relative to uninduced controls within man et al. 1995; Arman et al. 1998; Kunath et al. 2005; 2 h of doxycycline induction. This was followed by greater Chazaud et al. 2006; Yamanaka et al. 2010; Grabarek than twofold up-regulation of Sox17 and Pdgfra 4 and 6 h et al. 2012; Kang et al. 2013; Niakan et al. 2013), we also after induction, respectively. induced transgene expression in the presence of exoge- We also followed the fate of pulse-induced cells for 48 h nous Fgf4 and heparin, which facilitates FGF receptor after they were returned to pluripotency maintenance me- binding. dium in the absence of doxycycline (Fig. 2D). Over the Although the induced cells lost their mES cell-like mor- 48-h period, cells induced from 6 to 12 h up-regulated phology, again, only Gata6 or Gata4 expression resulted in Sox17 expression, suggesting that Gata6 had initiated iXEN cell reprograming within the 6-d time period (Fig. the endogenous XEN program (Fig. 2E). Consistent with 1A,C; Supplemental Fig. S1C–E). Additionally, we ob- this, flow cytometry analysis of Nanog expression in served little difference in overall gene expression between Gata6 pulsed cells identified a gradual reduction of medi- the FGF-supplemented and LIF-deficient conditions, sug- an Nanog expression with increased Gata6 induction time gesting that exogenous FGF signaling does not enhance re- periods (Fig. 2F; Supplemental Fig. S3). This suggests that programing of mES cells to iXEN cells. In contrast Sox7-, the induction of Gata6 leads to the inhibition of Nanog in Sox17-, Hnf4a-, or Foxa3-induced cells appeared to spor- most cells. Importantly, similar analysis in the pulse- adically differentiate following LIF withdrawal. Despite chased cells reveals a similar shift and indicates that, by the down-regulation of Pou5f1, Sox2, and Nanog, these 8 h of exogenous Gata6 expression, most of the cells do cells did not exhibit XEN-like morphology and inconsis- not re-express Nanog (Fig. 2F; Supplemental Fig. S3). How- tently up-regulated some but not all ExEn genes within ever, we occasionally observed some cells that retained this time frame. Altogether, this suggests that Gata6 or Nanog expression (Fig. 2E). As heterogeneities in Nanog Gata4 induction is uniquely able to rapidly reprogram expression are known to exist within mES cell cultures mES cells to iXEN cells. For subsequent experiments, we (Chambers et al. 2007), this suggests that these cells likely chose to use Gata6 transgenic mES cells, as Gata6 mutant have a higher threshold of pluripotency gene expression to embryos exhibit an early PrE deficiency, and Gata6 is overcome. thought to lie upstream of Gata4 in the PrE transcriptional Stable iXEN cell lines were successfully derived from hierarchy (Morrisey et al. 1998; Koutsourakis et al. 1999; Gata6-expressing cells induced for 6–12 h and were main- Chazaud et al. 2006; Plusa et al. 2008; Artus et al. 2011; tained in the absence of doxycycline for >10 passages. Schrode et al. 2014). Furthermore, all inductions were per- qRT–PCR analysis confirmed that stable iXEN cells main- formed in the presence of serum and LIF, as the absence of tain XEN gene expression and do not re-express pluripo- LIF alone destabilizes pluripotency gene expression. tency genes (Supplemental Fig. S2B). We next sought to test the developmental potential of iXEN cells by generating chimera embryos. We injected unlabeled iXEN cells A short pulse of Gata6 induction is sufficient to perturb into B5/EGFP embryonic day 3.5 (E3.5) blastocysts that the mES cell state constitutively express EGFP (Hadjantonakis et al. 1998). We next sought to investigate the minimum temporal re- We then analyzed chimera contribution in E5.5–E6.5 quirement for Gata6 induction to affect mES cell gene ex- embryos (Fig. 2G,H). In the majority of chimera embryos pression. We induced exogenous expression for defined (20 of 24), iXEN cells contributed to either the extraembry- pulses of between 2 and 12 h and fixed the cells for immu- onic visceral or parietal endoderm, and we did not detect nohistochemistry immediately following doxycycline epiblast contribution. This confirms that, similar to previ- withdrawal (Fig. 2A). Flag and Gata6 protein expression ous studies using eXEN cells (Kunath et al. 2005), our was detectable by immunofluorescence and Western iXEN cells were also capable of successful XEN contribu- blot analysis 2–4 h following doxycycline addition (Fig. tion in chimera embryos. 2B; Supplemental Fig. S2A). Although induced cells re- To gain further insight into the developmental poten- tained mES cell morphology over the initial pulse period, tial of iXEN cells, we introduced single unlabeled iXEN we observed a clear effect on protein expression, with cells into B5/EGFP E2.5–E3.5 embryos via morula aggre- Nanog protein down-regulated 8–10 h following induction gation or blastocyst injection and analyzed chimera con- (Fig. 2B). tribution at the blastocyst stage (Fig. 2G,I; Supplemental To examine gene expression dynamics in more detail, Fig. S2C). The majority of stable iXEN cells contributed we performed qRT–PCR analysis between 2 and 12 h fol- to Sox17-expressing PrE cells in chimeras (25 of 38). Inter- lowing Gata6 induction (Fig. 2C). qRT –PCR amplifica- estingly, when we introduced single Gata6 12-h pulse or tion of the Flag region confirmed robust induction of 12-h pulse-chase cells, we found that while a similar num- exogenous Flag-tagged Gata6 by 2 h after doxycycline in- ber of chimera embryos had exclusive Sox17 expression, duction and more than twofold down-regulation of Nanog the 12-h pulse-chase cells had a lower contribution to

1242 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Figure 2. A short pulse of Gata6 induction initiates iXEN cell reprograming (A) Time line of induction experiments. Cells were pulsed for incremental 2-h time periods and, at the end of each period, were analyzed by either immunohistochemistry, qRT–PCR, or flow cytometry. (B) Immunofluorescence analysis for Flag (green), Gata6 (red), and DAPI (blue) merge or with Nanog (green), Sox17 (red), and DAPI (blue) merge in Gata6-induced cells immediately after doxycycline treatment for the defined periods. Bars, 20 μm. (C) qRT–PCR analysis for exogenous Flag-tagged Gata6 expression and selected pluripotency and endoderm transcripts in Gata6-overexpressing mES cells between 0 and 12 h of doxycycline induction. Relative expression is reflected as fold difference over uninduced mES cells normalized to Gapdh. Data are mean ± SEM of two biological replicates. (D) Time line of induction pulse-chase experiments. Cells were pulsed for incremental 2-h time periods and, at the end of each period, were switched into pluripotency medium for 48 h and then analyzed by immunohistochemistry or flow cytometry or switched into pluripotency medium to derive stable iXEN cells. (E) Immunofluorescence analysis for Nanog (green), Sox17 (red), and DAPI (blue) merge in Gata6-induced cells treated with doxycycline treatment for the defined periods and then switched into pluripotency medium for 48 h. Bars, 20 μm. (F) Flow cytometry analysis of Nanog expression at the defined time points in the pulse and pulse-chase cells. (G–I) Chimera contribution of unlabeled Gata6-induced cells injected into B5/EGFP embryos that constitutively express EGFP. (G) Summary of the number of cells injected, the stage of injection and dissection, and chimera contribution. (H) Embryonic day 5.5 (E5.5)–E6.5 post-implantation embryos with unlabeled iXEN cell contribution to the visceral (arrow) or parietal (arrowhead) endoderm. Representative images of chimeras with phase-contrast, DAPI (blue) nuclear staining, and embryo EGFP expression. (I) E3.5–E4 chimera blastocysts immunofluorescently analyzed for the expression of the PrE marker Sox17 (red), the epiblast marker Nanog (white), EGFP (green), and DAPI (blue) merge. Bars, 100 μm. Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al.

Nanog-expressing cells (Fig. 2G,I; Supplemental Fig. S2C). with doxycycline-induced expression of Gata6 (Fig. 3E, This suggests that while Gata6 is potent and highly effi- F). We confirmed loss of Pou5f1 expression by qRT – cient in initiating iXEN cell reprograming, a sufficient PCR (Fig. 3E) and Oct4 protein by Western blot analysis time interval is required to commit to a XEN program. (Fig. 3F). Despite the premature loss of Oct4, Gata6-in- To further unravel the mechanisms of Gata6-mediated duced cells still up-regulated endogenous Gata6, Sox17, reprograming, we chose to investigate the effect of Gata6 Gata4, Lama1, Col4a1, and Sparc and down-regulated induction over a 6-d (144-h) period, as we had previously Sox2 and Esrrb (Fig. 3E). This suggests that Gata6 induc- observed complete down-regulation of Nanog, Oct4, and tion bypasses a possible requirement for Oct4 in repro- Sox2 by this time point. We analyzed defined time points graming mES cells to iXEN cells in vitro. To further between 12 and 144 h of induction to evaluate morpholo- confirm this, we used an Oct4 conditional knockout gy and gene expression dynamics. From 24 h following mES cell line that induces trophectoderm differentiation Gata6 induction, mES cell colonies changed from a following doxycycline-dependent loss of Oct4 (Niwa domed to a flattened shape as cells migrated away from et al. 2000; data not shown). We used a lentivirus to over- the center of the colony and eventually became refractile, express the human GATA6 gene downstream from an rounded, and dispersed, similar to eXEN cells (Fig. 3A). EF1α promoter, which we confirmed successfully repro- qRT–PCR analysis confirmed that Nanog and Sox2 tran- grams wild-type mES cells to iXEN cells (Supplemental scripts were rapidly down-regulated within 12 h of doxy- Fig. S2D,E), similar to the doxycycline-inducible sys- cycline induction, with protein expression decreasing to tem. When we overexpressed GATA6 in the Oct4-null levels below detection within 24 and 36 h, respectively cells, we observed the emergence of XEN-like cells (Fig. 3B,C). Pou5f1 transcript and Oct4 protein displayed (Fig. 3G), consistent with the Pou5f1 knockdown cells. prolonged expression until 48 h after induction but were Western blot analysis confirmed that Oct4 protein was down-regulated by 96 h. undetectable following 12 h of doxycycline treatment The qRT–PCR analysis also showed >10-fold up-regula- (Fig. 3H). tion of ExEn genes between 12 and 48 h following doxycy- To determine whether Gata6-induced reprograming is cline treatment (Fig. 3B). Interestingly, despite up- dependent on the availability of endogenous Fgf4, we regulation of Gata4 and Sox17 transcripts within 12 h of used site-specific recombination to integrate our induc- Gata6 induction (Fig. 2C), their proteins were not detect- ible Gata6 transgene into Fgf4−/− mES cell lines (Cho able by Western blot until 24 h following doxycycline et al. 2012; Kang et al. 2013). To eliminate the possibility treatment (Fig. 3C). Given that Nanog, Esrrb, and Sox2 of signaling from exogenous FGFs, we initially grew the are down-regulated in the absence of detectable Gata4 or Fgf4−/− mES cells for three passages in serum-free medi- Sox17 protein, this suggests that Gata6 directly mediates um in the presence of an Erk and Gsk3 inhibitor together initial down-regulation of the pluripotency program. De- with LIF (2i+LIF), as had been previously described (Ying spite robust induction of exogenous Sox17 over the 144- et al. 2008). Interestingly, Gata6 induction in Fgf4−/− h period, these cells retained expression of Nanog and mES cells in basal serum-free medium in the absence Oct4 (Fig. 3D) and show delayed up-regulation of endo- of 2i+LIF or exogenous FGFs resulted in iXEN cell repro- derm factors compared with Gata6 (Fig. 1C). This is con- graming and the up-regulation of Gata4, Gata6, Sox7, sistent with recent findings that Sox17-expressing cells and Sox17 (Fig. 3I,J), consistent with recent observations only acquire XEN-like morphology and down-regulate in mES cells transiently transfected with Gata6 (Kang pluripotency factor expression after 12 and 18 d of induc- et al. 2013). Significantly, we also found that Oct4, tion, respectively (McDonald et al. 2014). Altogether, this Sox2, and Nanog were down-regulated independently of shows that Gata6 is uniquely able to rapidly down-regu- endogenous Fgf4 following Gata6 induction (Fig. 3J). To late the core components of the pluripotency gene regula- investigate the initial response to reprograming, we ana- tory network and promote an ExEn program even in lyzed the Fgf4−/− Gata6-induced cells at defined time conditions that favor mES cell maintenance. points between 12 and 144 h of induction by qRT –PCR (Supplemental Fig. S2F). These cells exhibited similar expression dynamics for pluripotency gene down-regula- Gata6 can induce reprograming of mES cells tion and ExEn gene up-regulation compared with Fgf4+/+ independently of Oct4 expression and Fgf4 signaling − − Gata6-induced cells (Fig. 3B) or Fgf4 / Gata6-induced Oct4 has been shown to be required for Fgf4-mediated PrE cells treated with exogenous FGF and heparin (Sup- specification within the mouse embryo (Frum et al. 2013; plemental Fig. S2F). Fgf4−/− iXEN cell lines were main- Le Bin et al. 2014). Although Gata6 expression is initiated tained for >10 passages in the absence of exogenous in Oct4 or Nanog mutant embryos, the subsequent ab- Gata6 without loss of gene expression (Supplemental sence of Sox17 and Gata4 suggests that PrE formation is Fig. S2G) or iXEN cell morphology (Fig. 3I), demon- compromised (Frankenberg et al. 2011; Frum et al. 2013; strating that there appears to be no requirement for a Le Bin et al. 2014). Given the persistent expression of feedback loop up-regulating Fgf4 to reinforce Gata6- Pou5f1/Oct4 following Gata6 induction, we sought to in- mediated reprograming. Moreover, given the absence vestigate the interplay between Gata6, Oct4, and Fgf4 dur- of phosphorylated Erk (Fig. 3J), it is unlikely that Erk ing mES cell reprograming. signaling is required. In all, we found that neither To investigate the requirement for Oct4, we intro- Fgf4 nor Oct4 is required for Gata6-mediated repro- duced shRNAs directed against Pou5f1 simultaneously graming of mES cells to iXEN cells.

1244 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently reprograms diverse cell types

Figure 3. Gata6 induction results in dynamic changes in cell morphology and gene expression even in the absence of Oct4 and Fgf4. (A) Representative phase-contrast images of Gata6-overexpressing mES cells at defined time points from 0 to 144 h of doxycycline treatment. Bars, 100 μm. (B) qRT –PCR analysis for selected pluripotency and endoderm transcripts in Gata6-overexpressing mES cells between 0 and 144 h of doxycycline induction. Relative expression is reflected as fold difference over uninduced mES cells normalized to Gapdh. Data are mean ± SEM of three biological replicates. (C) Western blot for selected proteins in Gata6-overexpressing cells from 0 to 144 h of doxycycline induction. A representative Actin loading control is included as a reference. Endogenous (endo) and exogenous (exo) Gata6 bands are indicated. (D) Western blot for selected proteins in Sox17-overexpressing cells from 0 to 144 h of doxycycline induction. A representative Actin loading control is included as a reference. (E) qRT –PCR analysis for selected pluripotency and endoderm transcripts following 72 h of shRNA knockdown of Pou5f1 during Gata6 induction compared with scrambled control shRNA. Data are mean ± SEM of five distinct shRNA constructs and two biological replicates. (∗∗∗) P < 0.001. (F) Western blot for selected proteins in Pou5f1 knockdown cells at the time points indicated. A representative Actin loading control is included as a reference. (G) Phase-contrast images of Oct4 conditional knockout cells following 6 d in the absence or presence of exogenous Gata6 induction. Bars, 20 μm. (H) Western blot for Oct4 protein in Oct4 conditional knockout cells at the time points indicated in the presence or absence of exogenous HA-tagged GATA6. A representative Actin loading control is included as a reference. (I) Phase-contrast image of stable Fgf4−/− iXEN cells. Bars, 20 μm. (J) Western blot for selected proteins in Fgf4-null mES cells following 6 d of Gata6 induction in the absence or presence of Fgf4 (F) and heparin (H). A representative Actin loading control is included as a reference.

GENES & DEVELOPMENT 1245 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al.

Gata6 directly regulates multiple components ing after 36 h of induction in order to capture both positive of the pluripotency gene regulatory network and negative gene regulatory dynamics as observed in our as well as ExEn genes Western blot and qRT –PCR analyses (Fig. 3B,C). We identified 12,632 Gata6-bound regions enriched over the input To characterize the global transcriptional profile during control that were common between three biological repli- Gata6-mediated reprograming, we performed microarray cates, with a false discovery rate (FDR) of <0.01% (Supple- analysis at defined time points from 12 to 144 h after in- mental Table S2). We carried out de novo motif analysis duction. We included untreated mES cells to reflect the on the top 500 most significant Gata6-bound regions initial pluripotent state and eXEN cells as a reference for and, as expected, identified the canonical GATA motif ExEn gene expression. Additionally, we included Sox7-ex- as the most highly enriched (Fig. 4B). We determined the pressing cells at 144 h after induction to compare their binding distribution of Gata6 throughout the genome gene expression with Gata6-induced cells. To investigate and found significant enrichment <1000 base pairs (bp) up- gene expression dynamics, we performed K-means clus- stream of gene promoters compared with the whole ge- tering on the scaled microarray data to group differentially nome (P-value ≤ 3.7 × 10−65). This is in contrast to Gata6 expressed genes over the time course into 50 clusters (Fig. binding downstream from genes, which has no significant 4A; Supplemental Fig. S4). difference compared with the genome (P-value ≤ 0.277) We found that a number of functionally significant plu- (Fig. 4C). ripotency-associated genes were rapidly and persistently Importantly, we found Gata6 binding enrichment at down-regulated within 12 h of Gata6 induction, including genes encoding multiple components of the pluripotency Nanog, Sox2, Nr5a2, Klf2, and Nodal (cluster 25) (Fig. 4A; regulatory network, such as Esrrb, Lefty1, Nr5a2, Nanog, Supplemental Fig. S4; Supplemental Table S1). Additional and Pou5f1, whose expression is down-regulated during genes, including Esrrb, Dazl, Dlk1, Ecsit, and Jarid1b reprograming (Fig. 4D; Supplemental Fig. S5; Supplemen- (cluster 29), were more gradually down-regulated over tal Table S2). Gata6 was also enriched at a number of rap- the time course compared with cluster 25. Pou5f1, Lefty1, idly up-regulated ExEn-associated genes such as Gata4 Dppa4, and Dppa2 (cluster 18) were more persistently ex- and Pdgfra (Fig. 4D; Supplemental Table S2), further sug- pressed and eventually down-regulated, suggesting step- gesting that Gata6 directly regulates both pluripotency wise down-regulation of various nodes of the pluripotency and ExEn genes. Intriguingly, the GATA motifs within gene regulatory network. these regions share a high degree of sequence conservation Gata6 induction also up-regulated the expression of sev- between placental mammals in some but not all cases. We eral ExEn transcription factors, cell surface proteins, and also identified Gata6 enrichment upstream of Fgfr2, sug- basement membrane components in a step-wise manner gesting that Gata6 may be directly regulating FGF signal- (Fig. 4A; Supplemental Fig. S4; Supplemental Table S1). ing (Supplemental Fig. S5; Supplemental Table S2). Initial up-regulation of Gata6, Sox17, Sox7, Foxa1, To relate Gata6 binding to global gene expression dy- Foxa2, and Pdgfra (cluster 15) was followed by up-regula- namics, we compared the ChIP-seq analysis with our tion of Gata4, Hnf1b, Hnf4a, and the key cell surface and microarray cluster data set to identify the subset of basement membrane components Dab2 and Lama1 (clus- Gata6-bound genes that were dynamically regulated over ter 39), which are thought to confer an adherence dif- the microarray time course. We then performed gene on- ference to PrE cells (Gerbe et al. 2008; Niakan et al. tology (GO) analysis comparing the Gata6-bound subset 2010; Artus et al. 2011). Additional basement membrane with the total microarray data set using the GOrilla anal- components and ExEn genes, including Lamb2, Col4a1, ysis tool (Eden et al. 2009). Gata6-bound dynamically reg- Cited1, and Braf (cluster 38), were up-regulated later in ulated genes were associated with signal transduction and the time course. We did not identify key markers of ecto- regulation, DNA binding and transcriptional regulation, dermal (Nestin, Pax6, Sox1, and Sox3) or mesodermal cell adhesion and migration, and stem cell maintenance (Flk1, Hand1, Mixl1, Nkx2.5, and T ) lineages within our (Supplemental Table S3). To investigate whether particu- microarray data set, suggesting that Gata6 is specifically lar gene expression patterns correlated with GO, we iden- inducing an endoderm fate. In contrast, Sox7-expressing tified a number of clusters that were continuously up- cells after 144 h of induction largely retained gene expres- regulated or down-regulated over the Gata6 induction sion patterns similar to mES cells (Fig. 4A; Supplemental time course and compared their GO term enrichment pat- Fig. S4). Importantly, while Sox7-expressing cells have terns. Down-regulated genes (clusters 3, 6, 9, 18, 19, 20, 23, up-regulated some genes associated with XEN cell func- 24, 25, 29, 34, and 46) were associated with protein bind- tion, such as Sall4 (cluster 33) (Lim et al. 2008), as before, ing, sequence-specific DNA binding, and biosynthetic pro- they maintained expression of pluripotency factors (clus- cesses (Supplemental Table S3). In contrast, genes that ters 18, 25, and 29) and did not up-regulate endoderm-asso- were continuously up-regulated (clusters 11, 15, 30, 38, ciated genes and basement membrane proteins to the same 39, 40, and 47) were enriched for membrane components, extent as Gata6-induced cells (clusters 15, 28, and 39). endoderm formation, and protein glycosylation (Supple- Given the rapid changes in gene expression dynamics mental Table S3). observed in our microarray analysis, we hypothesized Importantly, our ChIP-seq and time-course transcrip- that Gata6 may directly regulate both pluripotency tome analysis revealed genes whose expression is also and ExEn genes. We performed ChIP followed by high- rapidly down-regulated and that cluster with known pluri- throughput sequencing (ChIP-seq) analysis of Gata6 bind- potency factors. Given that Gata6 directly regulates a

1246 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently reprograms diverse cell types

Figure 4. Gata6 enrichment near both pluripotency and endoderm genes. (A) Line plots of selected up-regulated or down-regulated clusters that contain key pluripotency or endoderm genes. Plots are based on mean scaled expression values from microarray analysis of Gata6-overexpressing cells from 12 to 144 h of doxycycline treatment. Uninduced mES cells, eXEN cells, and Sox7-overexpressing cells after 144 h of doxycycline treatment were also included. Genes were grouped into 50 clusters using K-means clustering according to normalized gene expression values scaled across time points. The trajectories of scaled expression values for individual genes in each cluster across time are shown as gray lines, while the purple lines correspond to the cluster mean. (B) MEME de novo motif analysis of the top Gata6-bound regions following ChIP-seq (ChIP followed by high-throughput sequencing) analysis. (C) Comparison of Gata6-binding distribution (light blue) at selected regions relative to the genomic average (dark blue). (∗∗∗) P < 0.001. (D) ChIP-seq binding profiles showing Gata6 enrichment near pluripotency factors Lefty1 and Esrrb and endoderm genes Gata4 and Pdgfra (dark green). The input control profile (IP) is included for comparison (light green). Representative ChIP-seq binding profile of three biological replicates.

GENES & DEVELOPMENT 1247 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al. number of known pluripotency factors, these additional ExEn genes were not identified among the Nanog, Oct4, genes may also function to maintain pluripotency. One and Sox2 target gene sets. Previous studies have shown such candidate is Etv5 (cluster 25), whose expression has that, in addition to its function in pluripotent cells also been associated with mES cells (Zhou et al. 2007), downstream from Nanog (Festuccia et al. 2012), Esrrb but whose function has not yet been tested in this context. knockdown or overexpression affects endoderm gene ex- Consequently, our work may provide a useful resource to pression (Ivanova et al. 2006; Loh et al. 2006; Uranishi identify putative pluripotency or, conversely, endoderm et al. 2013). However, when we overexpressed an Esrrb factors. However, the absence of Gata6 enrichment near transgene (van den Berg et al. 2008; Uranishi et al. 2013) Sox2, whose expression is also rapidly down-regulated, concomitant with doxycycline-induced Gata6 expression, may suggest indirect repression, possibly due to destabiliz- this did not prevent the down-regulation of Nanog expres- ing alternative nodes of the pluripotency regulatory net- sion (Supplemental Fig. S6A) or iXEN-like cell differentia- work. Alternatively, Gata6 may function as a repressor tion (data not shown). Moreover, by immunofluorescence via a binding site located further away. analysis, we found that in mouse preimplantation embryos, Esrrb and Gata6 expression was coincident even after down-regulation of Nanog in Gata6-high PrE cells (Supple- Gata6 shares common gene targets and binding sites mental Fig. S6B). This suggests that Esrrb may not function with pluripotency factors to inhibit ExEn differentiation in this context or that alter- We next sought to determine whether Gata6 shares com- native factors may be required in tandem to block the endo- mon gene targets and binding sites with pluripotency fac- derm-promoting effect of Gata6. Knockdown of additional tors, which would suggest competition for pluripotency or pluripotency factors such as Prdm14 and Nr5a2 has been ExEn target gene regulation. Using spatial heat map analy- shown to up-regulate ExEn genes (Ma et al. 2011; McDo- sis, we compared Gata6-bound loci identified in our ChIP- nald et al. 2014). Indeed, when we compared Gata6-bound seq analysis with published genome-wide occupancy of loci with those occupied by Prdm14 in mES cells, we iden- Oct4, Sox2, Nanog, Klf4, and Esrrb in mES cells (Fig. 5A; tified extensive overlap of common gene targets (Fig. 5B; (http://bioinformatics.cscr.cam.ac.uk/ES_Cell_ChIP-seq_ Supplemental Table S4) as well as overlapping binding compendium.html; Martello et al. 2012). We also investi- sites (843 overlapping gene loci) (Fig. 5A,C). Consequently, gated overlap between Gata6 and pluripotency factor gene the greater degree of overlap with Gata6-bound sites may targets (Fig. 5B). reflect the dual role of both Gata6 and a pluripotency factor We found that Gata6-bound loci directly overlapped subset in driving their respective cell fates. with 136 Oct4, 190 Nanog, 390 Klf4, or 188 Sox2 gene To investigate whether the binding sites that we identi- loci (Fig. 5A). Intriguingly, some of these sites were present fied during Gata6 reprograming are maintained in eXEN near pluripotency genes, including Lefty1 (Supplemental cells, we performed ChIP-seq analysis of Gata6 occupancy Fig. S5), suggesting that Gata6 may directly compete in eXEN cell lines (Supplemental Table S5,S6). Of the 927 with pluripotency factors to antagonize the regulation of Gata6 gene targets in eXEN cells, 504 genes were also tar- some common gene targets. Interestingly, we identified gets of Gata6 at the 36-h post-induction time point (Sup- common loci at the distal enhancers of Pou5f1 and Nanog plemental Table S4). This includes Gata6 endoderm (Fig. 5C), which are known to be relevant for pluripotency target genes such as Gata4, Gata6, Sox7, and Lamc1. In- regulation (Kagey et al. 2010). However, these sites form a terestingly, we did not detect enrichment of Gata6 bind- small proportion of the total Gata6-bound loci. We found ing near many of the pluripotency target genes that we more extensive overlap between Gata6 and Esrrb (671 identified at the 36-h post-induction time point, with overlapping gene loci) (Fig. 5A), and shared loci were pre- the notable exception of Nr5a2. This demonstrates that sent in both pluripotency and ExEn genes (Fig. 5C). Nota- Gata6 binding in eXEN cells is distinct compared with bly, Gata6 and Esrrb were enriched at a common locus cells in transition and suggests that there may not be a re- upstream of the endogenous Gata6 promoter, suggesting quirement for Gata6 to actively repress pluripotency fac- possible competition for regulation. tor expression long after they are down-regulated. We also observed that genes such as Nr5a2 and Esrrb were bound by both Gata6 and one or more of the pluripo- Gata6 initiates an ExEn program in differentiated cells tency factors but that these sites did not directly overlap and hES cells (Supplemental Fig. S5). When we examined the shared gene target data set (Fig. 5B; Supplemental Table S4), we We also sought to determine whether Gata6 could repro- found that out of 6192 identified Gata6 target genes, gram cells other than mES cells. To investigate this, we 4195 were also targets of Esrrb, 2815 were also targets of used a pure culture of stable neural stem cells that were Klf4, 1364 were also targets of Sox2, 1616 were also targets previously shown to generate functional neurons that of Nanog, and 1104 were also targets of Oct4 (Fig. 5B), sug- contribute to the adult brain in mouse chimeras, without gesting that Gata6 may regulate the pluripotency network the formation of teratomas (Conti et al. 2005). This avoids both directly and indirectly. Curiously, we identified the possibility of residual pluripotent or partially differen- several key ExEn genes within the list of Gata6 gene tar- tiated cells, which may be present at early stages of direct- gets shared with Esrrb, including Gata6, Gata4, Sox17, ed differentiation protocols. We expressed GATA6 in Col4a1, Fgfr2, Pdgfra, and Sox7 (Supplemental Fig. S5; neural stem cells by lentiviral transduction in neural basal Supplemental Table S4). In contrast, most of these key medium and evaluated the identity of the cells 20 d after

1248 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently reprograms diverse cell types

Figure 5. Gata6 binds to loci occupied by pluripotency factors in mES cells. (A) Density heat maps of Gata6-binding peak intensity after 36 h of Gata6 induction, indicating direct overlap with Nanog, Oct4, Sox2, Klf4, Esrrb, or Prdm14 binding in mES cells within a 10-kb window centered at the transcription start site (TSS). Data for Nanog, Oct4, Sox2, Klf4, Esrrb, and Prdm14 were obtained from the Mouse ES Cell ChIP-Seq Compendium (http://bioinformatics.cscr.cam.ac.uk/ES_Cell_ChIP-seq_compendium.html; Martello et al. 2012). (B) Venn diagram indicating the overlap of Gata6-bound genes during reprograming compared with genes previously shown to be bound by Oct4, Sox2, Klf4, Esrrb, or Prdm14 in mES cells. (C) Binding profiles at the Pou5f1, Nanog, Gata6, and Foxa2 loci for Gata6 and the input control during reprograming compared with Nanog, Oct4, Sox2, Klf4, Esrrb, or Prdm14 in mES cells. induction (Fig. 6A). Despite their morphological re- (Fig. 6B) in the GATA6-overexpressing cells. Importantly, semblance to neurons, immunofluorescence analysis con- the preinduced cells lacked detectable expression of Oct4 firms robust induction of Gata6, Sox7, and Sox17 proteins and Nanog (Conti et al. 2005), which we confirmed was

GENES & DEVELOPMENT 1249 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al.

Figure 6. Gata6 initiates an ExEn program in mouse neural stem cells and hES cells. (A) Phase-contrast images of uninduced or GATA6- induced mouse neural stem cells. Bars, 50 μm. (B) Immunofluorescence analysis for Sox7, Sox17, Gata6, or Nestin (green) and Oct4 or Nanog (red) with DAPI (blue) merge in GATA6-induced mouse neural stem cells after 20 d of induction. Bars, 50 μm. (C) qRT –PCR analysis of mouse neural stem cells for HA-tagged exogenous GATA6 and selected endoderm transcripts (Gata6, Sox7, Sox17, Col4a1, Lama1, Dab2, and Foxa3) 20 d following transduction. Data are mean ± SEM of two replicates. (D) Immunofluorescence analysis for OCT4 or GATA6 (green) and SOX17 (red) with DAPI (blue) merge in hES and human iPS (hiPS) cells in pluripotent culture conditions. Bars, 50 μm. (E) Representative phase-contrast images of GATA6-transduced hES cells 6 d after GATA6 induction compared with uninduced controls in pluripotency (mTeSR) or differentiation (KSR) medium. Bars, 50 μm. (F) Principal component analysis using RPKM (reads per kilobase per million mapped reads)-normalized RNA sequencing (RNA-seq) data from biological replicates of uninduced and GATA6-induced (+G) cells in KSR (K) or mTeSR (M) medium. (G) Heat maps showing the hierarchical clustering of uninduced and GATA6-induced (+G) cells in KSR (K) or mTeSR (M) medium using RPKM-normalized RNA-seq data. Expression levels plotted on a high-to-low scale (purple–white–green). (H) DESeq analysis indicating genes significantly differentially expressed in the uninduced control versus GATA6-induced cells in both the KSR and mTeSR conditions. The median gene expression and log2 fold change (FC) difference in expression are noted. (I) qRT –PCR analysis of H1 and H9 hES cells for HA-tagged exogenous GATA6 expression and selected pluripotency (POU5F1, NANOG, and SOX2) and endoderm (GATA6, SOX7, SOX17, and FOXA2) transcripts 6 d following transduction. Data are mean ± SEM of two to three replicates. (J) Immunofluorescence analysis of OCT4, NANOG, GATA4, SOX17, SOX17, and GATA6 (all green) and HA (red) with DAPI (blue) merge in GATA6-induced H9 hES cells 5 d following doxycycline treatment. Bars, 50 μm.

1250 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently reprograms diverse cell types also undetectable in the GATA6-induced cells (Fig. 6B). In tandem, we engineered GATA6 doxycycline-induc- We observed low levels of detectable Nestin expression ible hES cells by lentiviral transduction of a tetracycline/ in these cells (Fig. 6B), suggesting that an additional doxycycline-inducible HA-tagged GATA6 transgene. hES time interval may be required to stabilize the XEN cell cells in pluripotency medium treated with doxycycline program and fully overcome the neural stem cell state. exhibited a morphological switch similar to the GATA6 qRT –PCR analysis confirmed that the GATA6-induced virally transduced cells (Fig. 6E; data not shown). Impor- cells up-regulated the XEN factors Gata6, Sox7, Sox17, tantly, the GATA6 doxycycline-induced cells have Col4a1, Lama1, Dab2, and Foxa3 (Fig. 6C). In all, the abil- down-regulated OCT4 and NANOG in most cells (Fig. ity of Gata6 to promote reprograming to iXEN-like cells 6J). Moreover, the induced cells have up-regulated the ex- does not appear to be restricted solely to mES cells. pression of the HA-tagged GATA6, SOX7, SOX17, and We next sought to investigate the broader reprograming GATA4 proteins (Fig. 6J). While exogenous GATA6 initiat- potential of Gata6. We observed SOX17 and GATA6-coex- ed reprograming of hES cells to iXEN-like cells, the hetero- pressing cells within hES and iPS cell cultures maintained geneity in endoderm protein induction suggests that an in pluripotency conditions (Fig. 6D). This is reminiscent additional time interval, culture condition, or factor may of the Sox17-expressing XEN-committed cells that we ob- be needed to generate self-renewing human iXEN cell served previously in mES cell cultures (Niakan et al. 2010) lines. Nevertheless, this suggests that GATA6/Gata6 is and suggests that hES cell may also have the potential to sufficient to overcome a number of distinct cell states to be converted to XEN cell lines. We then transduced drive iXEN-like cell reprograming. hES cells in both pluripotency (mTeSR) and differentiation-promoting (KSR) conditions to determine whether Discussion GATA6 expression is sufficient to drive iXEN cell reprograming from hES cells. Significantly, as stable human We show that Gata6 functions to reprogram a number of XEN cell lines have yet to be established, no morphologi- cell types into iXEN-like cells. In the context of mES cal benchmark exists. cell reprograming, it does so by rapid and potentially di- GATA6-transduced hES cells exhibited a morphology rect repression of the pluripotency gene regulatory net- that is distinct from hES cell colonies even in conditions work coupled with activation of an endoderm gene that otherwise favor their pluripotency (Fig. 6E). We next program. What distinguishes Gata6 from other endoderm compared the global transcriptional profile of GATA6- transcription factors that we tested is the speed with transduced versus untransduced hES cells by RNA se- which it acts to induce a cell fate switch in the absence quencing (RNA-seq). We initially used principal compo- of selection. Indeed, most reprograming, including iPS nent analysis (PCA), which demonstrates that the cells, can take several days of selective culture. As the in- GATA6-induced samples cluster together irrespective of duction of Sox17 takes >2 wk to down-regulate pluripo- the basal medium and were transcriptionally distinct tency (McDonald et al. 2014), this suggests that indirect from untransduced cells (Fig. 6F). We also used an indepen- mechanisms eventually lead to XEN conversion, perhaps dent method of hierarchical clustering, which again dem- via Gata6. It would therefore be interesting to determine onstrates that, independent of the basal medium, the whether Sox17 can induce XEN reprograming in the ab- GATA6-induced cells cluster together and are distinct sence of Gata6. This seems unlikely given that Gata6 mu- from the untransduced cells (Fig. 6G). Notably, GATA6- tant mES cells fail to initiate cXEN cell conversion in transduced hES cells up-regulated a numberof extraembry- growth factor-mediated conditions, in contrast to Sox17 onic and/or pan-endoderm factors, including GATA6, mutant ES cells that initiate but fail to maintain cXEN GATA4, SOX17, SOX7, FOXA2, PDGFRA, COL4A1, cells (Cho et al. 2012). COL4A2, LAMA1, HNF4a, DAB2, and SPARC (Fig. 6G, Investigating the mechanisms of Gata6-mediated iXEN H; Supplemental Tables S5, S6). Significantly, we con- cell reprograming may provide insights into the genetic firmed that the GATA6-induced cells down-regulated plu- hierarchy involved in ExEn development in vivo (Fig. 7). ripotency factors, including NANOG, POU5F1, SOX2, In mouse embryos, Gata6 expression is initiated in the ab- PRDM14, FGF2, FOXD3, DPPA4, and ZSCAN10. qRT– sence of Oct4/Nanog-mediated Fgf4 signaling, but down- PCR analysis confirmed the up-regulation of endogenous stream Gata4 and Sox17 expression is compromised GATA6, SOX17, SOX7, and FOXA2 transcripts between (Frankenberg et al. 2011; Frum et al. 2013; Le Bin et al. 10-fold and 1000-fold and the down-regulation of the ex- 2014; Schrode et al. 2014). Our results suggest that induc- pression of NANOG, SOX2, and POU5F1 (Fig. 6I). Howev- tion of Gata6 can up-regulate Gata4 and Sox17 in the ab- er, expression ofgenes associated withalternativelineages, sence of Fgf4 in vitro, consistent with recent findings including FLK4, TBX3, CDX2, GATA3, SMAD6, EOMES, (Kang et al. 2013). Ectopic expression of Gata6 has also HAND1, and TUBB3, suggests that the GATA6-induced been shown to restore endoderm differentiation in ES cells hES cells may not be fully reprogramed to stable iXEN cells lacking the FGF signaling adaptor Grb2 (Wang et al. 2011). (Fig. 6I). Moreover, although we clonally passaged the We also show that Gata6 positively regulates itself as well GATA6-transduced hES cells more than three times and as Fgfr2, Gata4, and Sox17. This suggests that, in the em- they maintained their morphology for >1 mo, they could bryo, Gata6 may require a feedback loop via Fgf4/Fgfr2 sig- not be maintained indefinitely, suggesting that alternative naling to reinforce its own expression to achieve a certain conditions or factors may be required to derive stable hu- threshold, which subsequently triggers the expression of man iXEN cells. Gata4 and Sox17. This is consistent with the insufficiency

GENES & DEVELOPMENT 1251 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al.

shown). Interestingly, we identified Gata6 binding upstream of the Nanog promoter and within an intron of Esrrb. One possibility is to use the emerging RNA-guided CRISPR–Cas nuclease system (Cong et al. 2013; Mali et al. 2013) to mutagenize these endogenous sites to interrogate not only these putative Gata6 regulatory loci but also those of other pluripotency factors and endoderm targets. Remarkably, Gata6 drives iXEN-like cells from mouse neural stem cells, showing that it is a broad inducer of re- Figure 7. Model of the hierarchy of gene activity during iXEN reprograming. It is surprising that Gata6 can overcome programing. Here we show that the requirement for Oct4/Nanog intrinsic programs within these cell types. As mES cells during development to reinforce PrE development via FGF signal- have a characteristic open chromatin state, Gata6 may ing (A) can be bypassed in vitro with the induction of Gata6 (B), which can potently induce mES-to-iXEN cell reprograming in have fewer roadblocks to directly bind to regulatory ele- the absence of Oct4 or Fgf4. Gata6 can bind to and rapidly induce ments to control gene expression. However, in neural the expression of downstream elements of an ExEn gene regulato- cells, it would be surprising if all endoderm target genes ry network (including Gata4 and Sox17). Gata6 can also simulta- remained readily accessible for Gata6 direct regulation. neously inhibit the expression of core and peripheral components Therefore, one possibility is that Gata6 may be function- of the pluripotency gene regulatory network. In all, the dual func- ing as a pioneering transcription factor in these contexts, tion of Gata6 as a repressor and activator potently drives mES-to- exposing otherwise closed heterochromatic regions, as iXEN cell reprograming. has been shown for FoxA2 and Gata4 (Zaret and Carroll 2011). Given the pleiotropic function of Gata6 in promoting endoderm- and mesoderm-derived cell types, of exogenous Fgf4 to restore Sox17 expression in Gata6 it is also surprising that there is only one reprograming mutant embryos (Schrode et al. 2014). This may also ex- outcome. Further characterization of Gata6-mediated re- plain the colocalization of Nanog and Gata6 in vivo, programing in several cellular contexts would allow in- whereby a threshold of Gata6 expression needs to be terrogation of the relationship between transcription reached in order to overcome pluripotency and specify factors, signaling, and epigenetics in driving cell state the PrE. Gata6 overexpression in vitro likely exceeds transitions. this threshold, thereby leading to down-regulation of plu- Induction of SOX7 or SOX17 has been previously re- ripotency and up-regulation of ExEn genes, thus allowing ported to drive XEN-like or definitive endoderm-like cells, iXEN reprograming to proceed in the absence of Fgf4. In- respectively, from hES cells (Seguin et al. 2008). However, ducing high levels of Gata6 expression in Fgf4 mutant these cells cannot be maintained indefinitely in culture, mouse embryos would be one approach to test this hy- and, significantly, SOX7-orSOX17-expressing hES cells pothesis. Alternatively, Gata6 levels could be fine-tuned retain pluripotency gene expression (Seguin et al. 2008). in vitro to determine whether Gata6-low Fgf4 mutant GATA6 induction is able to both inhibit the pluripotency cells fail to induce Gata4 and Sox17. Lowering the dose program and promote ExEn gene expression, suggesting of doxycycline in our inducible system has been shown that, in this context, stable human XEN cells may have to reduce the penetrance and therefore the percentage of the potential to be established. Given the potency of the cells inducing expression rather than the quantitative lev- doxycycline-inducible system in initiating iXEN repro- els within a given cell (Beard et al. 2006). This apparent gramming in hES cells, it would be interesting to de- discrepancy between the in vivo requirement for Fgf4 sig- termine whether alternative culture conditions could naling in the PrE may also be the result of the presence of effectively capture stable human iXEN cells. Recent tran- signaling pathways that facilitate the destabilization of scriptomic analysis of human embryos (Yan et al. 2013) pluripotency in vitro or differences between ES cells and may lead to the identification of signaling pathways that early inner cell mass cells within the blastocyst (Boroviak may be important to stabilize GATA6-induced human et al. 2014). XEN cell lines. Together, this demonstrates that Gata6 One of the advantages of our analysis of global Gata6 is a versatile and potent reprograming factor that can act binding during the transition of mES cells to iXEN cells alone to drive a cell fate switch from diverse cell types. is that it revealed that Gata6 may function simultaneously as both an activator and repressor of genes during re- Materials and methods programing but not in eXEN cells. It has been suggested that other GATA transcription factors, such as Gata1, Culture conditions for pluripotent stem cell lines also have a dual activator and repressor role (Yu et al. and transcription factor induction 2009). However, it is unclear precisely how Gata6 func- mES cells were maintained on mouse embryonic fibroblast tions to regulate both sets of target genes and whether co- (MEF)-coated pregelatinized tissue culture plates (Corning) in se- factors, chromatin, or other epigenetic mechanisms may rum and 10 ng/mL LIF. Additional medium components are listed influence this decision. The analysis of sequences sur- in Supplemental Table S4. For details of the generation of in- rounding Gata6-bound loci has not yet revealed motifs ducible mES cell lines, see the Supplemental Material. Induction that consistently distinguish Gata6-bound down-regulat- of mES cells was performed in pluripotency maintenance me- ed genes from Gata6-bound up-regulated genes (data not dium using doxycycline at a final concentration of 1 µg/mL.

1252 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently reprograms diverse cell types

Doxycycline (1 µg/mL) was also used to generate Oct4-null cells ChIP-seq from ZHBTc4 mES cells (Niwa et al. 2000). H9 and H1 hES cells Gata6-inducible mES cells were seeded at 1 × 104 cells per square (WiCell) were cultured in mTeSR1 (Stem Cell Technologies) centimeter and treated with 1 µg/mL doxycycline for 36 h prior to on Matrigel (BD Biosciences)-coated dishes. Lentiviral packaging harvesting. Immunoprecipitation was performed on 1 × 107 to 2 × was performed in HEK293T cells using Lipofectamine 3000 (Life 107 cells as described (Vokes et al. 2007) for three biological repli- Technologies) cotransfection of a plasmid containing an EF1α cates versus input samples. Sonication was performed using a promoter driving the expression of human HA-tagged GATA6 Misonix 4000 (28 cycles of 15 sec on and 45 sec off at an intensity and the puromycin resistance gene (AMSbio) together with pack- of 70%) with a microtip probe (Misonix). The antibodies used are aging plasmids. Forty-eight hours after lentiviral transduction, listed in Supplemental Table S7. Libraries were prepared using cells were selected using 1 µg/mL puromycin (Sigma). the TruSeq ChIP sample preparation kit, and the resulting samples were sequenced using the Illumina Genome Analyzer II (Illu- qRT–PCR mina). Data will be deposited into Gene Expression Omnibus and released immediately after publication (GSE69323). Computa- RNA was isolated using TRI reagent (Sigma) and DNase I-treated tional analysis details are included in the Supplemental Material. (Ambion). cDNA was synthesized using a Maxima first strand cDNA synthesis kit (Fermentas). qRT –PCR was performed using Quantace Sensimix on an Applied Biosystems 7500 machine (Life Technologies Corporation). Primer pairs were previously pub- Acknowledgments lished (Molkentin et al. 1997; Fujikura et al. 2002; Niwa et al. We thank James Turner and Tiago Faial for advice and critical 2005; Brown et al. 2010a) or designed using Primer3 software. reading of the manuscript. We thank Zachary Gaber and Francois All primers are listed in Supplemental Table S6. Guillemot for the neural stem cell lines and media. We thank Lu- cas Baltussen and Sila Ultanir for the HEK293T cells. We are grate- Immunohistochemistry and imaging ful to Abdul Sesay and Leena Bhaw-Rosun at the Francis Crick Institute High-Throughput Sequencing Facility for their assis- Samples were fixed in 4% paraformaldehyde for 1 h or overnight tance with RNA-seq and ChIP-seq. We are grateful to Sarah John- at 4°C, permeabilized with 0.5% Tween in 1× PBS for 20 min, and son, Marta Miret, Nicolle Morey, and Lucia Goyeneche in the blocked with 10% FBS diluted in 0.1% Tween in 1× PBS for Procedural Services Section for their assistance with the chimera 1 h. Primary antibodies were diluted at 1:500 in blocking solu- experiments. We thank Clare Wise at the Stem Cell Facility, Don- tion, and samples were incubated overnight at 4°C rotating. Sec- ald Bell, and members of the Confocal and Image Analysis Labora- ondary antibodies were diluted at 1:300 in blocking solution, and tory. We thank Rudolf Jaenisch for targeting vectors, Austin samples were incubated for 1 h at room temperature, washed, and Smith for the neural stem cells and conditional Oct4 knockout covered with 0.1% Tween in 1× PBS containing DAPI Vecta- mES cells, and Tadayuki Akagi for the Esrrb expression plas- Shield mounting medium (Vector Laboratories). A list of the anti- mid. H.J. is supported by National Institutes of Health grant bodies used is in Supplemental Table S7. Images were taken on R01HG006841 and Maryland Stem Cell Research Fund 2012- either an Olympus 1X71 microscope with Cell^F software (Olym- MSCRFE-0135-00. This work was supported by The Francis Crick pus Corporation), a Zeiss Axiovert 200M microscope with Axio- Institute, which receives its core funding from Cancer Research Vision release 4.7 software (Carl Zeiss Ltd.), or a Leica SP5 UK; the UK Medical Research Council (MC_UP_1202/9); and inverted confocal microscope (Leica Microsystems Ltd). the Wellcome Trust. In addition, this research was supported by the March of Dimes Foundation (FY11-436) to K.K.N. Western blot analysis

Whole-cell protein was extracted with CelLytic M reagent References (Sigma) supplemented with proteinase and phosphatase inhibi- tors (Roche). Thirty micrograms of protein per sample was re- Arman E, Haffner-Krausz R, Chen Y, Heath JK, Lonai P. 1998. solved on 12% SDS-PAGE gels and transferred to PVDF Targeted disruption of fibroblast growth factor (FGF) receptor membrane using a Bio-Rad Trans-Blot transfer system (Bio-Rad). 2 suggests a role for FGF signaling in pregastrulation mamma- Membranes were blocked in 5% skimmed milk or 5% BSA in lian development. Proc Natl Acad Sci 95: 5082–5087. TBS 0.1% Tween and incubated with primary antibody overnight Artus J, Piliszek A, Hadjantonakis AK. 2011. The primitive endo- at 4°C. Following washes in TBS 0.1% Tween, membranes were derm lineage of the mouse blastocyst: sequential transcription incubated with secondary antibody in 5% milk or 5% BSA for factor activation and regulation of differentiation by Sox17. 1 h at room temperature. Proteins were visualized using the Dev Biol 350: 393–404. Pierce ECL Western blotting substrate (Thermo). Antibodies Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R. 2006. Effi- used are listed in Supplemental Table S7. cient method to generate single-copy transgenic mice by site- specific integration in embryonic stem cells. Genesis 44: 23–28. Microarray analysis Boroviak T, Loos R, Bertone P, Smith A, Nichols J. 2014. The abil- Total RNA was isolated as above and DNase I-treated (Ambion). ity of inner-cell-mass cells to self-renew as embryonic stem RNA quality was assessed on a eukaryote total RNA Nano series cells is acquired following epiblast specification. Nat Cell II (Agilent Technologies) and then processed on an Agilent 2100 Biol 16: 516–528. Bioanalyzer using the RNA electrophoresis program. All RNA Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP, samples were amplified using the Total Prep 96 RNA amplifica- Guenther MG, Kumar RM, Murray HL, Jenner RG, et al. tion kit (Ambion). Samples were hybridized to Illumina 2005. Core transcriptional regulatory circuitry in human em- MouseWG-6_V2 expression BeadChip arrays (Illumina, Inc.) Bio- bryonic stem cells. Cell 122: 947–956. logical triplicates were collected for each sample. Computational Brown K, Doss MX, Legros S, Artus J, Hadjantonakis AK, Foley analysis details are included in the Supplemental Material. AC. 2010a. Extraembryonic endoderm (XEN) stem cells

GENES & DEVELOPMENT 1253 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Wamaitha et al.

produce factors that activate heart formation. PLoS One 5: stem cells is induced by GATA factors. Genes Dev 16: e13446. 784–789. Brown K, Legros S, Artus J, Doss MX, Khanin R, Hadjantonakis Futaki S, Hayashi Y, Emoto T, Weber CN, Sekiguchi K. 2004. AK, Foley A. 2010b. A comparative analysis of extra-embryon- Sox7 plays crucial roles in parietal endoderm differentiation ic endoderm cell lines. PLoS One 5: e12016. in F9 embryonal carcinoma cells through regulating Gata-4 Capo-Chichi CD, Rula ME, Smedberg JL, Vanderveer L, Parma- and Gata-6 expression. Mol Cell Biol 24: 10492–10503. cek MS, Morrisey EE, Godwin AK, Xu XX. 2005. Perception Gerbe F, Cox B, Rossant J, Chazaud C. 2008. Dynamic expression of differentiation cues by GATA factors in primitive endo- of Lrp2 pathway members reveals progressive epithelial differ- derm lineage determination of mouse embryonic stem cells. entiation of primitive endoderm in mouse blastocyst. Dev Dev Biol 286: 574–586. Biol 313: 594–602. Chambers I, Silva J, Colby D, Nichols J, Nijmeijer B, Robertson Grabarek JB, Zyzynska K, Saiz N, Piliszek A, Frankenberg S, M, Vrana J, Jones K, Grotewold L, Smith A. 2007. Nanog safe- Nichols J, Hadjantonakis AK, Plusa B. 2012. Differential plas- guards pluripotency and mediates germline development. Na- ticity of epiblast and primitive endoderm precursors within ture 450: 1230–1234. the ICM of the early mouse embryo. Development 139: Chazaud C, Yamanaka Y, Pawson T, Rossant J. 2006. Early line- 129–139. age segregation between epiblast and primitive endoderm in Hadjantonakis AK, Gertsenstein M, Ikawa M, Okabe M, Nagy A. mouse blastocysts through the Grb2–MAPK pathway. Dev 1998. Generating green fluorescent mice by germline trans- Cell 10: 615–624. mission of green fluorescent ES cells. Mech Dev 76: 79–90. Chen WS, Manova K, Weinstein DC, Duncan SA, Plump AS, Pre- Hall J, Guo G, Wray J, Eyres I, Nichols J, Grotewold L, Morfopou- zioso VR, Bachvarova RF, Darnell JE Jr. 1994. Disruption of the lou S, Humphreys P, Mansfield W, Walker R, et al. 2009. Oct4 HNF-4 gene, expressed in visceral endoderm, leads to cell and LIF/Stat3 additively induce Kruppel factors to sustain em- death in embryonic ectoderm and impaired gastrulation of bryonic stem cell self-renewal. Cell Stem Cell 5: 597–609. mouse embryos. Genes Dev 8: 2466–2477. Hochedlinger K, Yamada Y, Beard C, Jaenisch R. 2005. Ectopic ex- Chen X, Xu H, Yuan P, Fang F, Huss M, Vega VB, Wong E, Orlov pression of Oct-4 blocks progenitor-cell differentiation and YL, Zhang W, Jiang J, et al. 2008. Integration of external signal- causes dysplasia in epithelial tissues. Cell 121: 465–477. ing pathways with the core transcriptional network in embry- Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, onic stem cells. Cell 133: 1106–1117. Schafer X, Lun Y, Lemischka IR. 2006. Dissecting self-renewal Cho LT, Wamaitha SE, Tsai IJ, Artus J, Sherwood RI, Pedersen in stem cells with RNA interference. Nature 442: 533–538. RA, Hadjantonakis AK, Niakan KK. 2012. Conversion from Kaestner KH, Hiemisch H, Schutz G. 1998. Targeted disruption mouse embryonic to extra-embryonic endoderm stem cells re- of the gene encoding hepatocyte nuclear factor 3γ results in reveals distinct differentiation capacities of pluripotent stem duced transcription of hepatocyte-specific genes. Mol Cell cell states. Development 139: 2866–2877. Biol 18: 4245–4251. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu Kagey MH, Newman JJ, Bilodeau S, Zhan Y, Orlando DA, van Ber- X, Jiang W, Marraffini LA, et al. 2013. Multiplex genome engi- kum NL, Ebmeier CC, Goossens J, Rahl PB, Levine SS, et al. neering using CRISPR/Cas systems. Science 339: 819–823. 2010. Mediator and cohesin connect gene expression and Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun chromatin architecture. Nature 467: 430–435. Y, Sanzone S, Ying QL, Cattaneo E, et al. 2005. Niche-inde- Kang M, Piliszek A, Artus J, Hadjantonakis AK. 2013. FGF4 is re- pendent symmetrical self-renewal of a mammalian tissue quired for lineage restriction and salt-and-pepper distribution stem cell. PLoS Biol 3: e283. of primitive endoderm factors but not their initial expression Davis RL, Weintraub H, Lassar AB. 1987. Expression of a single in the mouse. Development 140: 267–279. transfected cDNA converts fibroblasts to myoblasts. Cell 51: Koutsourakis M, Langeveld A, Patient R, Beddington R, Grosveld 987–1000. F. 1999. The transcription factor GATA6 is essential for early Eden E, Navon R, Steinfeld I, Lipson D, Yakhini Z. 2009. GOrilla: extraembryonic development. Development 126: 723–732. a tool for discovery and visualization of enriched GO terms in Kunath T, Arnaud D, Uy GD, Okamoto I, Chureau C, Yamanaka ranked gene lists. BMC Bioinformatics 10: 48. Y, Heard E, Gardner RL, Avner P, Rossant J. 2005. Imprinted Evans MJ, Kaufman MH. 1981. Establishment in culture of pluri- X-inactivation in extra-embryonic endoderm cell lines from potential cells from mouse embryos. Nature 292: 154–156. mouse blastocysts. Development 132: 1649–1661. Feldman B, Poueymirou W, Papaioannou VE, DeChiara TM, Le Bin GC, Munoz-Descalzo S, Kurowski A, Leitch H, Lou X, Goldfarb M. 1995. Requirement of FGF-4 for postimplantation Mansfield W, Etienne-Dumeau C, Grabole N, Mulas C, mouse development. Science 267: 246–249. Niwa H, et al. 2014. Oct4 is required for lineage priming in Festuccia N, Osorno R, Halbritter F, Karwacki-Neisius V, Nav- the developing inner cell mass of the mouse blastocyst. Devel- arro P, Colby D, Wong F, Yates A, Tomlinson SR, Chambers opment 141: 1001–1010. I. 2012. Esrrb is a direct Nanog target gene that can substitute Lim CY, Tam WL, Zhang J, Ang HS, Jia H, Lipovich L, Ng HH, for Nanog function in pluripotent cells. Cell Stem Cell 11: Wei CL, Sung WK, Robson P, et al. 2008. Sall4 regulates dis- 477–490. tinct transcription circuitries in different blastocyst-derived Frankenberg S, Gerbe F, Bessonnard S, Belville C, Pouchin P, Bar- stem cell lineages. Cell Stem Cell 3: 543–554. dot O, Chazaud C. 2011. Primitive endoderm differentiates Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, Chen X, Bourque G, via a three-step mechanism involving Nanog and RTK signal- George J, Leong B, Liu J, et al. 2006. The Oct4 and Nanog tran- ing. Dev Cell 21: 1005–1013. scription network regulates pluripotency in mouse embryonic Frum T, Halbisen MA, Wang C, Amiri H, Robson P, Ralston A. stem cells. Nat Genet 38: 431–440. 2013. Oct4 cell-autonomously promotes primitive endoderm Ma Z, Swigut T, Valouev A, Rada-Iglesias A, Wysocka J. 2011. Se- development in the mouse blastocyst. Dev Cell 25: 610–622. quence-specific regulator Prdm14 safeguards mouse ESCs Fujikura J, Yamato E, Yonemura S, Hosoda K, Masui S, Nakao K, from entering extraembryonic endoderm fates. Nat Struct Miyazaki Ji J, Niwa H. 2002. Differentiation of embryonic Mol Biol 18: 120–127.

1254 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently reprograms diverse cell types

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville stage differentiation of the extraembryonic endoderm in vitro. JE, Church GM. 2013. RNA-guided human genome engineer- J Cell Sci 120: 3859–3869. ing via Cas9. Science 339: 823–826. Shimosato D, Shiki M, Niwa H. 2007. Extra-embryonic endo- Martello G, Sugimoto T, Diamanti E, Joshi A, Hannah R, Oht- derm cells derived from ES cells induced by GATA factors ac- suka S, Gottgens B, Niwa H, Smith A. 2012. Esrrb is a pivotal quire the character of XEN cells. BMC Dev Biol 7: 80. target of the Gsk3/Tcf3 axis regulating embryonic stem cell Soudais C, Bielinska M, Heikinheimo M, MacArthur CA, Narita self-renewal. Cell Stem Cell 11: 491–504. N, Saffitz JE, Simon MC, Leiden JM, Wilson DB. 1995. Target- Martin GR. 1981. Isolation of a pluripotent cell line from early ed mutagenesis of the transcription factor GATA-4 gene in mouse embryos cultured in medium conditioned by teratocar- mouse embryonic stem cells disrupts visceral endoderm dif- cinoma stem cells. Proc Natl Acad Sci 78: 7634–7638. ferentiation in vitro. Development 121: 3877–3888. McDonald AC, Biechele S, Rossant J, Stanford WL. 2014. Sox17- Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem mediated XEN cell conversion identifies dynamic networks cells from mouse embryonic and adult fibroblast cultures by controlling cell-fate decisions in embryo-derived stem cells. defined factors. Cell 126: 663–676. Cell Rep 9: 780–793. Uranishi K, Akagi T, Sun C, Koide H, Yokota T. 2013. Dax1 asso- Molkentin JD, Lin Q, Duncan SA, Olson EN. 1997. Requirement ciates with Esrrb and regulates its function in embryonic stem of the transcription factor GATA4 for heart tube formation cells. Mol Cell Biol 33: 2056–2066. and ventral morphogenesis. Genes Dev 11: 1061–1072. van den Berg DL, Zhang W, Yates A, Engelen E, Takacs K, Bezstar- Morris SA, Teo RT, Li H, Robson P, Glover DM, Zernicka-Goetz osti K, Demmers J, Chambers I, Poot RA. 2008. Estrogen-relat- M. 2010. Origin and formation of the first two distinct cell ed receptor β interacts with Oct4 to positively regulate Nanog types of the inner cell mass in the mouse embryo. Proc Natl gene expression. Mol Cell Biol 28: 5986–5995. Acad Sci 107: 6364–6369. Vokes SA, Ji H, McCuine S, Tenzen T, Giles S, Zhong S, Longa- Morrisey EE, Tang Z, Sigrist K, Lu MM, Jiang F, Ip HS, Parmacek baugh WJ, Davidson EH, Wong WH, McMahon AP. 2007. Ge- MS. 1998. GATA6 regulates HNF4 and is required for differen- nomic characterization of Gli-activator targets in sonic tiation of visceral endoderm in the mouse embryo. Genes Dev hedgehog-mediated neural patterning. Development 134: 12: 3579–3590. 1977–1989. Niakan KK, Ji H, Maehr R, Vokes SA, Rodolfa KT, Sherwood RI, Wang Y, Smedberg JL, Cai KQ, Capo-Chichi DC, Xu XX. 2011. Ec- Yamaki M, Dimos JT, Chen AE, Melton DA, et al. 2010. Sox17 topic expression of GATA6 bypasses requirement for Grb2 in promotes differentiation in mouse embryonic stem cells by primitive endoderm formation. Dev Dyn 240: 566–576. directly regulating extraembryonic gene expression and indi- Yamanaka Y, Lanner F, Rossant J. 2010. FGF signal-dependent rectly antagonizing self-renewal. Genes Dev 24: 312–326. segregation of primitive endoderm and epiblast in the mouse Niakan KK, Schrode N, Cho LT, Hadjantonakis AK. 2013. Deriva- blastocyst. Development 137: 715–724. tion of extraembryonic endoderm stem (XEN) cells from Yan L, Yang M, Guo H, Yang L, Wu J, Li R, Liu P, Lian Y, Zheng X, mouse embryos and embryonic stem cells. Nat Protoc 8: Yan J, et al. 2013. Single-cell RNA-seq profiling of human pre- 1028–1041. implantation embryos and embryonic stem cells. Nat Struct Niwa H, Miyazaki J, Smith AG. 2000. Quantitative expression of Mol Biol 20: 1131–1139. Oct-3/4 defines differentiation, dedifferentiation or self-re- Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, newal of ES cells. Nat Genet 24: 372–376. Cohen P, Smith A. 2008. The ground state of embryonic stem Niwa H, Toyooka Y, Shimosato D, Strumpf D, Takahashi K, Yagi cell self-renewal. Nature 453: 519–523. R, Rossant J. 2005. Interaction between Oct3/4 and Cdx2 de- Yu M, Riva L, Xie H, Schindler Y, Moran TB, Cheng Y, Yu D, Har- termines trophectoderm differentiation. Cell 123: 917–929. dison R, Weiss MJ, Orkin SH, et al. 2009. Insights into GATA- Niwa H, Ogawa K, Shimosato D, Adachi K. 2009. A parallel cir- 1-mediated gene activation versus repression via genome- cuit of LIF signalling pathways maintains pluripotency of wide chromatin occupancy analysis. Mol Cell 36: 682–695. mouse ES cells. Nature 460: 118–122. Zaret KS, Carroll JS. 2011. Pioneer transcription factors: estab- Plusa B, Piliszek A, Frankenberg S, Artus J, Hadjantonakis AK. lishing competence for gene expression. Genes Dev 25: 2008. Distinct sequential cell behaviours direct primitive en- 2227–2241. doderm formation in the mouse blastocyst. Development 135: Zhang X, Zhang J, Wang T, Esteban MA, Pei D. 2008. Esrrb acti- 3081–3091. vates Oct4 transcription and sustains self-renewal and pluri- Schrode N, Saiz N, Di Talia S, Hadjantonakis AK. 2014. GATA6 potency in embryonic stem cells. J Biol Chem 283: levels modulate primitive endoderm cell fate choice and tim- 35825–35833. ing in the mouse blastocyst. Dev Cell 29: 454–467. Zhou Q, Chipperfield H, Melton DA, Wong WH. 2007. A gene Seguin CA, Draper JS, Nagy A, Rossant J. 2008. Establishment of regulatory network in mouse embryonic stem cells. Proc endoderm progenitors by SOX transcription factor expression Natl Acad Sci 104: 16438–16443. in human embryonic stem cells. Cell Stem Cell 3: 182–195. Zhou Q, Brown J, Kanarek A, Rajagopal J, Melton DA. 2008. In Shimoda M, Kanai-Azuma M, Hara K, Miyazaki S, Kanai Y, Mon- vivo reprogramming of adult pancreatic exocrine cells to den M, Miyazaki J. 2007. Sox17 plays a substantial role in late- β-cells. Nature 455: 627–632.

GENES & DEVELOPMENT 1255 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells

Sissy E. Wamaitha, Ignacio del Valle, Lily T.Y. Cho, et al.

Genes Dev. 2015, 29: Access the most recent version at doi:10.1101/gad.257071.114

Supplemental http://genesdev.cshlp.org/content/suppl/2015/06/24/29.12.1239.DC1 Material

References This article cites 70 articles, 29 of which can be accessed free at: http://genesdev.cshlp.org/content/29/12/1239.full.html#ref-list-1

Creative This article, published in Genes & Development, is available under a Creative Commons Commons License (Attribution 4.0 International), as described at License http://creativecommons.org/licenses/by/4.0/.

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the top Service right corner of the article or click here.